Rolling circle replication exploits the fact that replication of circular nucleic acid molecules is essentially an endless process producing repeated copies of the circle—this is how prokaryotic genomes are replicated in nature.
The research variant of the reaction employs linear oligonucleotides which are shaped into circles, typically by ligating the two ends together after they have been put in proximity by hybridisation to a ligation template. Subsequently, these circles may be copied in a rolling circle replication. This reaction is usually initiated by adding a primer to the closed circle, but as pointed out in WO 97/20948, it may equally well be initiated from the ligation template (the primary hybridisation target).
The reaction in the research setting is often referred to as a rolling circle amplification (RCA), though this should, strictly speaking, be reserved for situations where the rolling circle product is further amplified by a hyperbranch or DNA cascade reaction (WO 97/19193 and WO 97/20948).
The rolling circle product may remain a string of tandemly repeated copies of the circle, or may be reduced to monomers by digestion with a restriction enzyme or a ribozyme. This basic process is described in the patent literature (e.g. WO 98/38300) and scientific papers (e.g. Dahl F et al., Proc Natl Acad Sci USA. 101(13), 4548-53 (2004)).
Detection of specific nucleic acids by hybridisation in cell and tissue samples is of significant interest both for research and diagnostic purposes. Originally this was done by hybridisation of labelled DNA or RNA probes to the specimens (in situ hybridisation). However, the molecular resolution (ability to detect variations in the hybridisation target) and sensitivity of this approach is insufficient for a number of purposes.
A modified approach was therefore introduced, where unlabelled linear short (oligonucleotide) probes are employed to induce the synthesis of labelled DNA at the hybridisation site. This so-called PRimed IN Situ technique (PRINS, Koch et al. 1989, and many subsequent publications) provides improved resolution and sensitivity through the better discrimination among target sequences obtained with the short probes, as well as signal amplification from the site specific DNA synthesis, but only works for certain hybridisation targets.
A strategy to improve the performance of the technique was therefore proposed in WO 97/20948. According to this strategy, circular oligonucleotide probes are used in place of the linear probes, and the site-specific DNA synthesis is primed from the hybridisation target using the circle probe as template for the DNA synthesis, whereby the hybridisation target becomes extended with numerous tandemly repeated copies of the complementary sequence to the DNA circle. These copies may then be detected either through the incorporation of labelled nucleotides during the DNA synthesis, or through subsequent hybridisation to the tandem repeat. This localised production of recognizable DNA by rolling circle replication is referred to as rolling circle PRINS in the following part of this section.
Localised detection of nucleic acid molecules by rolling circle PRINS requires that the synthesis reaction is efficiently retained at the site where these molecules originally were. This can be obtained by using a free 3′-end of the target nucleic acid molecule as primer for the DNA polymerase, enabling it to initiate the rolling circle replication of the circle probe. In DNA, such ends may already be available as the result of biological processes breaking the DNA in vivo (Andersen C L. et al. Chromosome Res. 10(4), 305-12 (2002)) or as preparation artefacts. Such “naturally occurring” 3′-ends were employed in WO 97/20948. Alternatively, suitably placed 3′-ends can be generated using nucleases to prepare the target DNA for the reaction, if the DNA is digested with an endonuclease 3′ of the target site. If the resulting end is not right next to where the circle hybridises, the target DNA is digested with an exonuclease or a polymerase having exonuclease activity to recess the 3′-end until a point where it can prime the rolling circle process. These steps can be performed before, during or after the hybridisation of the circle to the target DNA as described in WO 99/49079 and in Larsson C. et al. Nature Methods 1, 227-32 (2004). WO 02/50310 mentions that not only DNA but also RNA may be detected by rolling circle DNA synthesis (in solution, on slides and in paraffin sections p. 11, I.6). However, no indications are given for the preparation of the target RNA for the rolling circle process, and the tool provided for the DNA detection (restriction digestion) is not applicable to RNA targets. Additionally, the process for DNA detection requires the addition of separate rolling circle primers, and, since no difference is emphasized, this must apply to the RNA detection as well.
Thus, in conclusion, a rolling circle DNA synthesis based approach specific to the target primed detection of RNA targets was neither provided in WO 97/20948 nor mentioned in WO 02/50310.
Rolling circle detection of RNA was also suggested in WO 99/49079 and WO 01/77383. These patent applications elaborated on the basic concept of performing rolling circle detection on RNA targets by providing optimised reaction conditions for the formation of a circle probe through ligation on an RNA template, and suggest that breakage of the target molecule may be obtained with either RNase H or RNase A. However, despite optimising the conditions for probe formation, the yield of DNA circles under the optimised conditions was still significantly lower than the yield obtained when circles are formed on a DNA template. As for the digestion with RNase A, it provides random cleavage of the RNA target, and not the wanted targeted cleavage. Targeted cleavage may be obtained by digesting the RNA with RNase H, which specifically cleaves the RNA component in DNA/RNA hybrids, leading to RNA degradation exclusively at sites where the circle probe is located. Unfortunately, this degradation leads to the dislocation of the probe from its hybridisation target, so that it can no longer report on the location of that target (Koch, unpublished observation). An efficient RNA detection version of the target primed rolling circle PRINS previously described for DNA detection is thus still not published.
US2003/0087241 discloses small single stranded circular oligonucleotide templates for synthesis of oligonucleotides, preferably RNA oligonucleotides. The circular oligonucleotides further comprise means for converting synthesised multimer into monomers; such means may e.g. be a selfcleaving ribozyme. The purpose of the method disclosed is efficient, low-cost and large scale synthesis of oligonucleotides. A detection method was not contemplated.
Thus, in spite of the recent progress of the rolling circle technology there is still a need for improvements of the technique, notably to provide detection of nucleic acid sequences other than double stranded DNA sequences containing appropriately positioned restriction sites. In particular, further developments of the technique are needed, rendering site specific cleavage of single stranded nucleic acid sequences, including RNA and single stranded DNA, possible, in order to allow detection of such sequences by target primed rolling circle PRINS.